Three extraction methods for determination of lipids in fish meal: Evaluation of a hexane/isopropanol method as an alternative to chloroform-based methods

Three different extraction methods were compared for the determination of lipids in both oxidised and antioxidant-stabilised brown fish meal. The three methods under investigation were those of Smith-Ambrose-Knobl using chloroform/methanol/water, Bligh and Dyer also using chloroform/methanol/water but monophasic, and Hara and Radin using hexane/isopropanol. The fat content, determined gravimetrically, was significantly lower (P < 0.05) when hexane/isopropanol was used for lipid extraction from both oxidised and antioxidant-stabilised fish meal, than when either of the chloroform systems was used. Determination of lipid classes in the lipid extracts using silica gel thin-layer chromatography on Chromarods-SIII, with flame ionisation detection by latroscan, suggested that the lower lipid recovery by the hexane/isopropanol method may be due to a less effective extraction of the more polar lipid classes. including oxidation products, present in brown fish meal.     

Concentration of polar MFGM lipids from buttermilk by microfiltration and supercritical fluid extraction

Buttermilk contains the milk fat globule membrane (MFGM), a material that possesses many complex lipids that function as nutritionally valuable molecules. Milk-derived sphingolipids and phospholipids affect numerous cell functions, including regulating growth and development, molecular transport systems, stress responses, cross membrane trafficking, and absorption processes. We developed a two-step method to produce buttermilk derivative ingredients containing increased concentrations of the polar MFGM lipids by microfiltration and supercritical fluid extraction (SFE). These processes offer environmentally benign alternatives to conventional lipid fractionation methods that rely on toxic solvents. Firstly, using a ceramic tubular membrane with 0.8-micron pore size, we evaluated the cross flow microfiltration system that maximally concentrated the polar MFGM lipids using a 2n factorial design; the experimental factors were buttermilk source (fresh, or reconstituted from powder) and temperature (50 degrees C, and 4 degrees C). Secondly, a SFE process using supercritical carbon dioxide removed exclusively nonpolar lipid material from the microfiltered buttermilk product. Lipid analysis showed that after SFE, the product contained a significantly reduced concentration of nonpolar lipids, and a significantly increased concentration of polar lipids derived from the MFGM. Particle size analysis revealed an impact of SFE on the product structure. The efficiency of the SFE system using the microfiltration-processed powder was compared much more favorably to using buttermilk powder.     

Phospho- and sphingolipid distribution during processing of milk, butter and whey

Phospho‐ and sphingolipids (SPHs) were monitored upon processing of raw milk into skimmed milk, cream, butter, buttermilk, fresh cheese, acid whey, anhydrous milk fat and butterserum (=the aqueous phase of butter). These products were analysed on polar lipids, fat and dry matter and corresponding balances were calculated. It was found that polar lipids were preferentially enriched in aqueous phases like skimmed milk, buttermilk and butterserum. Significant differences in relative SPH content were observed. Tangential filtration and thermocalcic aggregation of the acid whey successfully recovered the polar lipids. With 11.5% of polar lipids on a dry matter base, representing 28.4% of the milk polar lipids and only 0.9% of the milk mass, butterserum was found to be most suitable for further purification. This could result in a food (ingredient) with enhanced technological and nutritional properties, as recent studies reveal health‐improving capacities of phospho‐ and SPHs.     

高效液相色谱法分析原料乳和奶粉中三聚氰胺

建立了二极管阵列检测器-高效液相色谱测定原料乳及奶粉中三聚氰胺的方法。奶粉样品经三氯乙酸超声提取,乙酸铅沉淀蛋白,三氯甲烷除去脂肪和非极性杂质,PROELUTPXCSPE阳离子交换固相萃取小柱进一步除去样品基质干扰,氨化甲醇洗脱。实验对色谱分离条件进行了优化,三聚氰胺的质量浓度在1.0～100.0μg/mL范围内与色谱峰面积呈良好的线性关系,回归方程为y=89.763x-113.72,相关系数为R2=0.9988。加标回收率为90.5%～105.0%,相对标准偏差(RSD)小于4.0%。该方法简单、快速,结果准确可靠。     

Optimization of Solvent Extraction and Direct Transmethylation Methods for the Analysis of Egg Yolk Lipids

The composition of egg yolk lipids differs greatly from most other food lipids, as it combines high contents of triglycerides and phospholipids, as well as a high proportion of cholesterol. The lipids are organized in lipoproteins and are dispersed in aqueous phase. Classical extraction methods using chloroform and methanol have not been optimized for such a matrix, therefore quantitative extraction yields of egg lipids can be inaccurate. In this study, the original method of Bligh and Dyer is evaluated and adapted for egg lipids. Furthermore, a direct extraction/derivatization method is presented. Solvent extraction resulted in a lipid yield of 31.8 ± 0.89% and total fatty acid yield was 257 ± 2 mg/g yolk whereas the direct method yielded 250 ± 7 mg/g yolk. Gas chromatographic analyses confirmed that lipid recovery was complete for triglycerides and phospholipids as well.     

Use of polar aprotic solvents to release membranes from milk lipid globules

In the search for alternatives to physical methods for release of membranes from milk lipid globules, aqueous solutions of the polar aprotic solvents dimethyl sulfoxide and dimethyl formamide were found to cause release of milk fat globule membrane. Membranes released with polar, aprotic solvents displayed some differences in polypeptide, enzymatic, and lipid composition in comparison with membranes released by churning. However, all polypeptide, enzymatic, and lipid constituents measured in membranes released by churning were also present in membranes released with dimethyl formamide and dimethyl sulfoxide. Antigenic and Concanavalin A binding activity of polypeptides was retained during exposure to dimethyl sulfoxide or dimethyl formamide. The solvent method was effective in releasing membrane from lipid globules of cow, rat, and guinea pig milks. This solvent method was rapid and minimized losses attendant with physical methods for release of milk fat globule membrane.     

Lipid rafts in the bovine milk fat globule membrane revealed by the lateral segregation of phospholipids and heterogeneous distribution of glycoproteins

This study reveals the lateral organisation of the milk fat globule membrane (MFGM). Using confocal laser scanning microscopy (CLSM) and a lipid soluble molecule, an exogenous phospholipid and two lectins as fluorescent probes we located triacylglycerols in the core of fat globules and investigated the organisation of the polar lipids and glycoproteins of the MFGM, in situ in milk. Lipid rafts corresponding to the lateral segregation of sphingolipids in liquid-ordered phases surrounded by liquid-disordered domains composed by the glycerophospholipids were observed in the MFGM. These lipid rafts which correspond to rigid sphingolipid-rich domains have a circular shape at room temperature. CLSM experiments revealed that glycoproteins and glycolipids are heterogeneously distributed around fat globules and that they are not located in the lipid rafts. The characterisations performed by in depth thin sectioning of fat globules and in dynamic as a function of time revealed chemical and structural heterogeneities in the MFGM. Schematic 3D and 2D representations of the MFGM are proposed and discussed. The physiological and nutritional consequences of the lateral organisation of polar lipids and glycoproteins in the MFGM are discussed but remain to be elucidated.     

Properties,analysis and purification of milk polar lipids

The phospholipids and sphingolipids in milk are gaining interest due to their nutritional and technological qualities. Sphingolipids and their derivatives are highly bioactive compounds with anti-cancer, bacteriostatic and cholesterol-lowering properties. Several low-value process streams of the dairy industry contain considerable amounts of polar lipid and exert potential for further purification. This review deals with the structure of the main dairy polar lipids, their origin and molecular arrangement, their occurrence in raw milk and other dairy products, their methodology of analysis and purification and their nutritional and technological properties.     

Effect of solvent to sample ratio on total lipid extracted and fatty acid composition in meat products within different fat content

The effect of solvent to sample ratio on total extracted lipids and fatty acid (FA) composition in meat products with different fat contents was evaluated. Total lipids were extracted according to the Folch et al. (1957) method, using a 20:1 ratio of chloroform:methanol (2:1, v/v) to sample (A), and also testing the solvent:sample ratio of 10:1 (B). Higher amounts of total lipids and total FA from neutral lipids were obtained using the A ratio, which could be due to an insufficient chloroform:dry-weight sample proportion which could be insufficient for solubilizing the total amount of lipids. In the polar lipid fraction, the total amount of FA was higher using the B rather than the A ratio, which may be caused by the higher volume of added water when using A than B. When studying the FA composition of different lipid fractions, the volume of both the solvent and the water for total lipid extraction should be considered.     

EXTENDED VALIDATION OF A SIMPLIFIED EXTRACTION AND GRAVIMETRIC DETERMINATION OF TOTAL FAT TO SELECTED FOODS

A simplified method for chloroform/methanol extraction and gravimetric determination of total fat previously published for diet composites was tested on additional foods, including milk, cheese, fried snack foods, nuts/seeds, salad dressings, baked goods, meat, fish/shellfish and fruits/vegetables. Homogenized food composites and certified reference materials (CRMs) were analyzed using the existing method involving, briefly, orbital shaking of a subsample with chloroform/methanol/buffer in specific proportions, then recovery of total lipid in the chloroform extract. For meat, fish, shrimp, cheese and fried plantains, total fat recovery was low and/or variability was high with the unmodified assay versus results from other standard total fat methods (e.g., ether extraction, acid hydrolysis) or to CRM assigned values. Reducing the sample size or adding a homogenization step during extraction resulted in relative standard deviations of < 3% for all matrices and also values consistent with those generated by other standard total fat methods and within assigned ranges for total fat in most CRMs.

PRACTICAL APPLICATIONS

The method allows for routine batch analysis of multiple samples of a range of food types with minimized hands-on time, using commonly available laboratory equipment. The resulting total lipid extract can be subdivided for analysis of various specific lipid analytes as well as for gravimetric measurement of total fat.     

Characterization of the Lipid Fraction in Lamb Meat: Comparison of Different Lipid Extraction Methods

The aim of this study was to evaluate the performance of four different extraction methods commonly used to quantify the intramuscular lipid content in meat: the Association of Analytical Chemists (AOAC) method (reference method) and methods based on the use of a solvent mixture with different polarities, such as chloroform–methanol described by Folch et al. (J Biol Chem 226:497, 1957) and Christie (1989) or hexane–isopropanol described by Hara and Radin (Anal Biochem 90:420, 1978). The following parameters were taken into account: lipid content; relative proportions of neutral and polar lipids; fatty acid composition of total, neutral, and polar lipid fractions; and phospholipid composition. The use of a combination of solvents with different polarities (Hara–Radin, Folch, and modified Folch methods) was more effective in extracting intramuscular lipids than the use of a single solvent (AOAC, reference method). The Hara–Radin method provided a cleaner lipid extract with a significantly higher content of total fatty acids than that obtained with the Folch and modified Folch methods. The lower polarity of the hexane–isopropanol mixture allowed us to obtain an extract richer in neutral lipids (triglycerides and diglycerides) and thus in saturated and monounsaturated fatty acids. The percentage distribution of individual fatty acids in the neutral lipid fraction was generally not affected by the extraction method adopted, while lipid obtained with both the Hara–Radin and Folch methods had a polar fraction with a higher percentage of polyunsaturated fatty acids. The use of the Hara–Radin method provided a polar fraction with less nonlipid material and lower phospholipid degradation.     

Buffalo milk fat globules and their biological membrane: in situ structural investigations

Milk fat globules and their surrounding biological membrane (the MFGM) are not well understood despite the importance of these milk components in human nutrition and the role of fat globules in determining the properties of dairy products. The objectives of this study were to investigate these unique colloidal assemblies and the microstructure of the MFGM in buffalo milk, which is the second largest global source of dairy products. In-situ structural investigations were performed at room temperature using confocal microscopy with multiple fluorescent probes (Nile Red, Rh-DOPE, the lectin WGA-488). Microscopic observations showed cytoplasmic crescents around fat globules and the heterogeneous distribution of glycosylated molecules and polar lipids with the occurrence of lipid domains. The lipid domains in the buffalo MFGM appear to form by the segregation of lipids with a high phase transition temperature (e.g. sphingomyelin and saturated phosphatidylcholine molecular species) and cholesterol resulting in a gel phase or a Lo phase forming circular domains. The structure of the buffalo MFGM results from a non-random mixing of components, consistent with observations for other species. Structural heterogeneities of the MFGM could affect the processability of buffalo fat globules and the bioavailability of milk lipids.     

Rapid Method for the Quantitative Extraction and Simultaneous Class Separation of Milk Lipids

A method is described for the isolation of lipids from milk, cream, or buttermilk. Lipid isolation is accomplished by solvent elution of a column containing a mixture of the milk product, anhydrous sodium sulfate, and Celite 545. Total lipids are isolated by elution with a 90:10 mixture of dichloromethane:methanol. Alternatively, lipids may be separated into a neutral lipid fraction by sequential elution of the column with dichloromethane and a polar fraction by elution with a 90:10 mixture of dichloromethane:methanol. The proposed method was compared with the traditional Roese-Gottlieb method for a series of milk samples with fat content ranging from < 1 to 37%. Fat determinations for a series of five milk types by the dry column and Roese-Gottlieb methods were significantly different for whole raw milk and buttermilk, while means for phospholipids were significantly different for all milk types tested except buttermilk.     

Bioactive Sphingolipids are Constituents of Soy and Dairy Products

ABSTRACT: Dietary sphingolipids have gained attention because of their possible ability to inhibit colon cancer. The purpose of this study was to quantify sphingolipids in soy and dairy products, 2 food groups that are associated with protection against colon cancer. Sphingolipids were extracted with chloroform and methanol, and quantified by HPLC. The concentrations of total sphingolipids (nmol/g of dry weight) were: full fat soy flakes, 609; soy flour, 610; isolated soy protein, 210; nonfat dry milk, 203; Swiss cheese, 167; and yogurt, 138. Most sphingolipids were complexed with sphingosine as the predominant sphingoid base backbone. The results also indicate that total lipid content is not a good predictor of the relative content of sphingolipids in soy and dairy products.     

The influence of temperature and pressure factors in supercritical fluid extraction for optimizing nonpolar lipid extraction from buttermilk powder

The milk fat globule membrane, readily available in buttermilk, contains complex lipids claimed to be beneficial to humans. Phospholipids, including sphingolipids, exhibit antioxidative, anticarcinogenic, and antiatherogenic properties and have essential roles in numerous cell functions. Microfiltration coupled with supercritical fluid extraction (SFE) may provide a method for removing triacylglycerols while concentrating these nutritionally valuable lipids into a novel ingredient. Therefore, SFE as a method for phospholipid concentration needs to be optimized for triacylglycerol removal in buttermilk. The SFE conditions were assessed using a general full factorial design; the experimental factors were pressure (15, 25, and 35 MPa) and temperature (40, 50, and 60°C). Particularly interesting is that only triacylglycerols were removed from buttermilk powder. Little to no protein loss or aggregation was observed compared with the untreated buttermilk powder. Calculated theoretical values showed a linear increase for lipid solubility as pressure, temperature, or both were increased; however, experimental values showed nonlinearity, as an effect of temperature. In addition, the particular SFE parameters of 35 MPa and 50°C displayed enhanced extraction efficiency (70% total lipid reduction).     

南极磷虾脂质提取方法的比较

实验分别比较了氯仿甲醇、正己烷和95%乙醇三种不同溶剂以及超临界CO2提取的南极磷虾脂质成分差异,为南极磷虾脂质的研究提供一定基础。结果表明,氯仿甲醇提取的脂质得率最高,为19.21%±0.59%。正己烷提取的得率最低,仅为14.24%±0.38%,甘油三酯和胆固醇含量最高,分别为46.98%±1.00%和3.68%±0.30%,但对极性脂的提取效果不理想,磷脂含量仅为19.20%±3.45%。95%乙醇提取的脂质磷脂含量35.59%±2.69%,不饱和脂肪酸中EPA和DHA含量是最高的,占总脂肪酸含量的41.13%,且95%乙醇安全性高,价格低廉,适合大规模工业化生产,可以作进一步的探讨。超临界CO2萃取技术作为脂质的一种温和提取手段,得到的脂质基本成分与氯仿甲醇近似,虽然安全性更高,但是超临界设备成本较高,不适于中小型企业工业化生产。     

牛乳极性脂质的提取工艺优化及成分鉴定

为更好开发利用牛乳中的乳极性脂质,本文采用氯仿-甲醇浸提法对酪乳粉中的极性脂质进行提取研究。以磷脂含量为指标,通过单因素试验对氯仿-甲醇体积比、液料比、提取温度及提取时间四个影响牛乳极性脂质提取的因素参数进行优化,后利用响应面法筛选最优提取条件,并采用薄层层析对所得样品进行了分析鉴定。结果表明,氯仿-甲醇比为2:1（v/v, %）最优的料液比为1:4（v/v, %）,提取温度20 ℃,提取时间10 min为极性脂质的最优提取条件,在此条件下测得实际提取磷脂含量为（1.647±0.010）mg/g,且存在磷脂酰胆碱、磷脂酰乙醇胺、磷脂酰丝氨酸、神经鞘磷脂等极性脂质。本工艺旨在为乳极性脂质的提取及开发提供研究基础。     

Lipid composition and structural characteristics of bovine,caprine and human milk fat globules

Milk fat is widely accepted to be the major nutrient in human milk. Commercial infant formulas are usually based on mammalian milk such as bovine or caprine milk, but the differences in milk fat globules (MFGs) between human, bovine and caprine milk remain unclear. We showed that saturated fatty acid content was higher in bovine and caprine MFGs (>60%) than in human MFG (<40%), but that content of the unsaturated fatty acids C18:2 in human MFG was >7 times higher than in bovine and caprine MFGs. The cholesterol content of human milk was ∼20% higher than that of bovine and caprine milk. Triacylglycerol molecular species and polar lipids also differed between bovine, caprine and human MFGs. Confocal laser scanning microscopy images of MFGs revealed that the shape of the liquid-ordered domains varied by species.     

Validation of a rapid milk fat separation method to determine the fatty acid profile by gas chromatography

An improved rapid method for separating lipids from milk to determine the fatty acid composition using 2 centrifugations at room temperature (20°C) was compared with the ISO-IDF reference procedure based on solvent extraction. The new method is useful for research and routine quality control and has a number of advantages over the reference procedure—mainly no solvents are required and it saves time. Applicability of the rapid separation method was confirmed in fats with different physical characteristics from ewe and goat milk samples. Minor differences were found in the proportions of some fatty acids in the reference and centrifugation methods. Milk fat separated by centrifugation at room temperature did not differ in fatty acid composition from milk centrifuged at 4°C.     

Stability of Metabolically Conjugated Precursors of Meat and Milk Flavor Compounds in Various Solvents

The stability of selected metabolic conjugates (phenylglucuronide, phenylphosphate, and naphthylsulfate) was determined in model systems composed of water and various ratios (3:1, 1:1, 1:3) of selected solvents (hexane, chloroform, ethyl acetate, diethyl ether, or methanol) held at either 22°C or 40°C for 30 min under various pH conditions (pH 1.5, 3.2, or 6.5). Notable hydrolysis occurred only for the more polar solvents held in contact with acidic aqueous phases. Conditions were identified for minimizing hydrolysis of conjugates during extraction of fat and free alkylphenols from milk and meat products with diethyl ether. They were pH near neutral, short exposure time, near ambient temperature, presence of excess water, and saturation of aqueous phase with sodium chloride.